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BOR - Papers in Press, published online ahead of print August 17, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.042317
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BIOLOGY OF REPRODUCTION 73, 1269–1274 (2005)
DOI: 10.1095/biolreprod.105.042317
© 2005 by the Society for the Study of Reproduction, Inc.

Research Article

Analysis of Flagellar Bending in Hamster Spermatozoa: Characterization of an Effective Stroke

Masashi Kinukawa 2, Junko Ohmuro 3, Shoji A. Baba 3, Sunao Murashige 4, Makoto Okuno 5, Masao Nagata 2, and Fugaku Aoki 1 2

Department of Integrated Biosciences,2 Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan Department of Biology,3 Ochanomizu University, Tokyo 112-0816, Japan Department of Complexity Science and Engineering,4 Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8651, Japan Department of Life Sciences,5 Graduate School of Arts and Sciences, University of Tokyo, Tokyo 153-8902, Japan

ABSTRACT

The mechanism by which flagella generate the propulsive force for movement of hamster spermatozoa was analyzed quantitatively. Tracing points positioned 30, 60, 90, and 120 µm from the head-midpiece junction on the flagellum revealed that they all had zigzag trajectories. These points departed from and returned to the line that crossed the direction of progression. They moved along the concave side (but not the convex side) of the flagellar envelope that was drawn by tracing the trajectory of the entire flagellum. To clarify this asymmetry, the bending rate was analyzed by measuring the curvatures of points 30, 60, 90, and 120 µm from the head-midpiece junction. The bending rate was not constant through the cycle of flagellar bending. The rate was higher when bending was in the direction described by the curve of the hook-shaped head (defined as a principal bend [P-bend]) to the opposite side (R-bend). We measured a lower bending rate in the principal direction (R-bend to P-bend). To identify the point at which the propulsive force is generated efficiently within the cycle of flagellar bending, we calculated the propulsive force generated at each point on the flagellum. The value of the propulsive force was positive whenever the flagellum bent from an R-bend to a P-bend (when the bending rate was lowest). By contrast, the propulsive force value was zero or negative when the flagellum bent in the other direction (when the bending rate was higher). These results indicate that flagellar bending in hamster spermatozoa produces alternate effective and ineffective strokes during propulsion.

effective stroke, flagellar bending, gamete biology, sperm, sperm motility and transport

INTRODUCTION

Progressive movement of spermatozoa occurs through propulsive forces generated by flagellar bending, which is in turn caused by the sliding of microtubules in the flagellar axoneme. The flagellar axoneme of most eukaryotic flagella typically comprises a central pair of singlet microtubules surrounded by nine doublet microtubules (9 + 2 arrangement). Flagellar bending is generated by the sliding of adjacent doublet microtubules via the activity of dynein arm ATPase, which uses Mg2+-ATP as the substrate [1–2]. Beating consists of an undulating wave propagating from the base to the tip of the flagellum. According to Newton's third law of motion (law of action and reaction), a force generated by flagellar bending toward the direction of the tip would push the spermatozoa forward [3].

Although the mechanism regulating microtubule sliding in the flagellar axoneme has been studied extensively, little is known about the mechanism through which flagellar bending generates the propulsive force that moves spermatozoa. There are simulation models of the mechanism by which microtubule sliding generates flagellar bending [4–18]. There are also a few simulation models explaining how flagellar bending generates progressive motility [19–20], but the bending pattern of the flagellum in these models approximates a sine curve. These simulations cannot be applied to mammalian spermatozoa. In rodents, the bend angle is much steeper when the flagellum bends in the direction of the curve of the hook-shaped head of the spermatozoon than when it bends in the opposite direction [21–23]. Therefore, detailed analyses of flagellar bending are important for elucidating the regulation of spermatozoon motility.

To clarify the mechanism by which flagellar bending generates propulsive force in spermatozoa efficiently, we examined the process quantitatively in the hamster. We used hamsters as experimental animals, because the waveform is two-dimensional, making it easy to analyze flagellar bending using phase-contrast microscopy. Furthermore, the shape of the rodent spermatozoon head resembles a hook, which is useful for determining the direction of flagellar bending. Our analysis shows that flagellar bending is functionally, as well as geometrically, asymmetrical, in that it produces effective and ineffective strokes for movement alternately.

MATERIALS AND METHODS

Media Preparation

The medium used for in vitro culture of hamster spermatozoa consisted of 110 mM NaCl, 5.0 mM KCl, 2.4 mM CaCl2, 0.49 mM MgCl2, 0.36 mM NaH2PO4, 24.9 mM NaHCO3, 25 mM Hepes, 6.25 mM lactic acid, 0.125 mM sodium pyruvate, 0.5 mM hypotaurine, 5.0 mM glucose, 12 mg/ml BSA (fatty acid-free Fraction V; Sigma), 100 U/ml penicillin, and 0.1 mg/ml streptomycin [24]. The pH of the medium was adjusted to 7.4.

Spermatozoa Preparation

Sexually mature male Syrian hamsters (Mesocricetus auratus) were raised and maintained in a light-controlled room (12L:12D) at constant temperature (22°C ± 1°C). The hamsters were killed by chloroform inhalation, and the cauda epididymis was removed promptly. After removing blood from the epididymal surface with physiological salt solution, the distal tubules were punctured in 5 to 10 places with an 18-gauge needle, and a mass of spermatozoa was squeezed out with forceps into a plastic Petri dish (35 x 10 mm) containing 5 ml of medium prewarmed to 37°C. The spermatozoa were incubated at 37°C under 5% CO2 in air. All of the procedures described herein were reviewed and approved by the University of Tokyo Institutional Animal Care and Use Committee, and were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.

Sample Preparation

Aliquots (14 µl) of the spermatozoa suspension were withdrawn for flagellar bending analysis after 10 min or 4 h of incubation. The suspension was diluted with medium to give a final concentration of approximately 2 x 106 spermatozoa per ml. A glass slide was warmed to 37°C. The suspension was placed on the glass slide and covered with an 18 x 24-mm coverslip to a depth of about 32 µm. As soon as the sample was prepared, photographic images were taken for analysis.

Analysis of Flagellar Bending

Flagellar bending of the spermatozoa was analyzed from images taken at 250 frames per second with a FASTCAM-Net high-speed camera (Photron). Phase contrast microscopy was used, and the exposure time for each image was 1/1000 s. The images obtained were recorded and analyzed using Bohboh (BohbohSoft) flagellar image analysis software. The curvature and angle between the tangent at each point on the flagellum and the direction of progression were measured as described previously [25–26]. The bending rate was measured using 120 successive frames of the image. The direction of each bend was determined by the direction of the hook-shaped projection of the head. A bend in the direction described by the curve of the hook-shaped head was defined as a principal bend (P-bend), and one in the opposite direction was defined as a reverse bend (R-bend). The curvatures in the principal and reverse bend were assigned plus and minus values, respectively. Points 30, 60, 90, and 120 µm from the head-midpiece junction were termed D30, D60, D90, and D120, respectively. The direction of progression of the spermatozoa was defined by a line connecting the outermost points in the zigzag trajectory of the head-midpiece junction.

Typical movement patterns of spermatozoa that had been incubated for 10 min or 4 h are shown in movie files (Supplemental Figures S1 and S2, respectively [available online at http://www.biolreprod.org/]).

Calculation of Propulsive Force

The propulsive force of the flagellum was calculated using the method of Gray and Hancock [19]. Motion at a low Reynolds number, such as the propulsion of spermatozoa, is regarded as directly proportional to the velocity of displacement and to the viscosity of the medium. Therefore, the propulsive forces exerted on a short element ({delta}s) in the flagellum are represented by the following values. The net propulsive force (dF) is


Formula

where CT is the coefficient of resistance acting tangentially, CN is the coefficient of resistance acting normally, and {theta} is the orientation of a short element to the direction of progression. The orientation was determined by measuring the angle between the tangent at each position of the flagellum and the direction of progression. The direction of progression was defined as above. The velocity from the longitudinal displacement (Vx) and the transverse displacement (Vy) are

Formula

Formula
where x is the x coordinate, y is the y coordinate, K is the frame number, and r is the frame rate (Hz).

The coefficients of resistance acting tangentially (CT) and normally (CN) to the surface of the element for a medium of known viscosity are

Formula


Formula
where µ is the viscosity of the medium, d is the radius of the flagellum, and {lambda} is the wavelength. CN is effectively twice the value of CT for very thin filaments, such as a flagellum [27]. The average wave length (as an index of wave length), determined by doubling the value derived from the length of the flagellum (180 µm; [28]) divided by the number of bends, was 75 µm and 110 µm in the spermatozoa incubated for 10 min and 4 h, respectively.

RESULTS

The trajectory was analyzed by tracing the entire flagellum to draw the flagellar envelope in each free-swimming spermatozoon. Figure 1 shows the flagellar envelope in a single beat cycle in a spermatozoon incubated for 4 h. Tracing the positions of D30, D60, D90, and D120 on the flagellar envelope revealed that all drew arcs (red lines in Fig. 1). When the entire flagellum was traced over several beat cycles, the flagellar envelope drew a trajectory that curved in the same direction as the curve of the head (Fig. 2A), and all of the points (D30–D120) drew zigzag trajectories on this envelope (red lines in Fig. 2A). When the direction of progression of the spermatozoa was defined by a line connecting the outermost points in the zigzag trajectory of the head-midpiece junction (blue arrows in Fig. 2B), the points D30–D120 all departed from and returned to a line crossing the direction of progression (making no forward progression), and the points moved along the concave edge of the flagellar envelope (making some forward progression; Fig. 2A). However, they did not move along the convex edge of the flagellar envelope, indicating no progression along this convex edge. This suggests that the propulsive force is generated asymmetrically during the cycle of flagellar bending. The difference between the P- and R-bends [23] is probably involved in this asymmetry. To address this possibility, the bending rate was analyzed, because it is an important factor in determining the propulsive force. In Figure 3A, the curvatures at points D30, D60, D90, and D120 on the flagellum are plotted against time, whereby the slope of the line indicates the bending rate. The slopes of the lines from the point of maximum value (MX) to that of minimum value (MN) (P-bend to R-bend) were always higher than those going from the points of MN to MX (R-bend to P-bend) at all points on the flagellum, except D30, for which the slopes were almost equal (Fig. 3B). These results demonstrate that the bending rate is not constant in the cycle, with highest rates when the flagellum bends in the P-bend to R-bend direction.


Figure 1
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  		FIG. 1. Trajectories of entire flagella in a single beat cycle of freely swimming spermatozoa. Spermatozoa that had been incubated for 4 h were analyzed. Images were taken at the rate of 250 frames per 4 seconds and the entire flagella are traced in a single beat cycle to draw the flagellar envelope. The starting image is that in which the angle of the R-bend was maximal for each point. The points 30, 60, 90, and 120 µm from the head-midpiece junction (D30, D60, D90, and D120, respectively) on the flagellum are indicated by red circles. The trajectories of these points are shown by the red line connecting successive points (red circles) on the flagellar envelope. The positions of the head-midpiece junction are indicated by black squares. Bar = 100 µm


Figure 2
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  		FIG. 2. Trajectories of whole flagella and the direction of progression in freely swimming spermatozoa. Spermatozoa that had been incubated for 4 h were analyzed. Images were taken at the rate of 250 frames/sec for 0.48 sec, during which time several beat cycles were recorded. The flagellar envelope (the trajectories of the entire flagellum) is indicated by the black lines. On the flagellar envelope, the trajectories of the points 30, 60, 90, and 120 µm from the head-midpiece junction (D30, D60, D90, and D120, respectively) on the flagellum (A) and that of the head-midpiece junction (B) are depicted by the red line. The flagellar envelope presented in B is identical to that presented in A. The direction of progression of the spermatozoa, which was defined by a line connecting the outermost points in the trajectory of the head-midpiece junction, is depicted by the blue arrows to the left of the trajectories (B). Three spermatozoa were analyzed with similar results, and a representative sample is shown. Bars = 100 µm


Figure 3
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  		FIG. 3. Analysis of the bending rate during a cycle of flagellar bending. A) Changes in the curvature at various points of the flagellum with time. Spermatozoa incubated for 4 h were photographed at a rate of 250 frames/sec (hence, the elapsed time between two adjacent frames was 1/250 sec). The curvatures were measured 30, 60, 90, and 120 µm from the head-midpiece junction on the flagellum (D30, D60, D90, and D120, respectively) for each time-point. Three spermatozoa were analyzed with similar results, and a representative sample is shown. B) The bending rates at various points on the flagellum. The bending rates were determined by measuring the slope from the point of maximum value (MX) to that of minimum value (MN) (P to R) and the slope from MN to MX (R to P) at points D30, D60, D90, and D120. This analysis was performed using more than three cycles of flagellar bending in 120 successive frames, and the data were averaged. Three spermatozoa were analyzed and the data are expressed as mean ± SEM (n = 3)

The propulsive force for each point on the flagellum was calculated using the formula proposed by Gray and Hancock [19]. Interestingly, the value of the propulsive force was positive whenever the flagellum bent in the direction from R-bend to P-bend (for which the bending rate was lower) and was zero or negative from P-bend to R-bend (for which the bending rate was higher) at all points on the flagella of spermatozoa that were incubated for 4 h (Fig. 4). Similar results were obtained for the spermatozoa incubated for 10 min, with only a few exceptions (data not shown). These results demonstrate that spermatozoa alternately produce effective and ineffective strokes for progressive movement.


Figure 4
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  		FIG. 4. Analyses of curvature and propulsive force. Spermatozoa incubated for 4 h were photographed at a rate of 250 frames/sec (hence, the elapsed time between two adjacent frames was 1/250 sec). The changes in curvature and propulsive force were examined 30, 60, 90, and 120 µm from the head-midpiece junction (D30, D60, D90, and D120, respectively) on the flagellum. The propulsive forces with positive values relative to the direction of movement are indicated by red dots, and those with negative values are designated by blue dots. The curvature data in this figure are identical to those shown in Figure 3. Three spermatozoa were analyzed, with similar results, and a representative sample is shown

DISCUSSION

We identified bending in the direction R-bend to P-bend as the effective stroke. In the switch-point hypothesis advocated by Satir [11] and Holwill and Satir [14], the active bundles of the dynein arms switch in an alternating fashion, generating alternating bending between P-bend and R-bend. It has been suggested that the bundle of microtubule doublets 4–5–6–7 is located on the same side as the curve of the hook-shaped head, and that the bundle of 9–1–2 microtubules is located on the opposite side (see Fig. 5) [29–30]. Given that the dynein arm attached to the doublet microtubule (n) pushes the next doublet (n + 1) in the direction from base to tip [31] with an exception that 5–6 microtubules are tightly connected to each other [32–34], the dynein arms attached to microtubules 6–7–8–9 should generate the effective stroke for propulsive force [35–37]. These reports suggest that the dynein arms attached to microtubules 6–7–8–9 and 1–2–3–4 are important in generating effective and ineffective strokes, respectively.


Figure 5
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  		FIG. 5. Diagram of a flagellar axoneme showing microtubule numbers. Note that microtubule doublets 5 and 6 are tightly connected to each other

Similar to flagellar bending in hamster spermatozoa, the ciliary beat also consists of two asymmetrical components, i.e., the power and return strokes [3]. A ciliary beat cycle consists of an effective stroke, in which the extended cilium makes an oarlike movement toward one side, and a recovery stroke, in which the cilium moves back from base to tip [38]. Although axoneme structures are the same in flagella and cilia (9 + 2 arrangement of microtubules), the active dynein arms responsible for the effective and recovery strokes in cilia are the opposite of those in hamster flagella; those attached to microtubules 1–2–3–4 and 6–7–8–9 in cilia are responsible for the effective and recovery strokes, respectively [37]. However, it is possible that the mechanism generating asymmetric microtubule sliding is similar in flagella and cilia, but the machinery regulating asymmetric sliding is simply arranged differently for the numbered microtubules. Supporting this hypothesis is the observation that the ciliary beat is arrested at the end of the recovery stroke by treatment with high concentrations of Ca2+ and calcium ionophore, A23187 [37]. In mouse spermatozoa, treatment with A23187 increased the asymmetry of flagellar bending in the direction of the R-bend, which is the end point of the recovery stroke [39–40]. Therefore, the dyneins sensitive to Ca2+ seem to be located in opposite positions in flagella and cilia. The machinery regulating the effective stroke may also be located in opposite positions in these two systems.

The bending rate was always slower in the direction from R- to P-bend than in the reverse direction (Fig. 3), and the effective stroke was always generated by the slower of the two (Fig. 4). These results suggest that the sliding of microtubules is regulated differently in the two directions. Indeed, Ca2+ affects flagellar bending asymmetrically; it increases bending in the direction of the R-bend [41–42]. The fact that the maximum value of the R-bend was always smaller than that of the P-bend (Fig. 3A) also suggests that the difference in elasticity (imparted by the difference in the structure of the flagellar axoneme) between the side of the R-bend and that of the P-bend is involved in the asymmetric flagellar bending. Because there are nine circumferential microtubule doublets and outer dense fibers, an odd number, their distribution is not equal between the bundles on the R-bend and P-bend sides, which may cause the difference in the elasticity of the flagellum in these two directions. The linking proteins, such as the nexin link and radial spoke, might also work asymmetrically. Therefore, the elasticity imparted by these complexes within the axoneme might play a role in generating the asymmetric effective stroke for progressive movement.

The effective stroke was observed in spermatozoa incubated for 10 min and in those incubated for 4 h. At both times, the bending rate was asymmetric and the propulsive force was generated when the flagellum bent from the direction of the R-bend to the P-bend. However, after 4 h of incubation the spermatozoa were hyperactivated, and it has been suggested that the propulsive force is increased in this case; the swimming speed is increased [23], and spermatozoa can swim in medium with high viscosity, whereas nonhyperactivated cells cannot [43]. When the effective stroke generates a propulsive force, amplitude is the most important element determining the propulsive force. It has been shown that the amplitude of bending is increased in hyperactivated spermatozoa [23]. We also confirmed that the amplitudes at points D60, D90, and D120 on the flagellum were higher in spermatozoa incubated for 4 h than in those incubated for 10 min; the amplitudes were almost equal among incubation times at D30 (unpublished results). Therefore, a greater amplitude seems to contribute to the larger propulsive force in hyperactivated hamster spermatozoa.

The Reynolds number is important in analyzing various types of flow. The power output derived from a wave is dependent on the Reynolds number. The Reynolds number at the size scale of humans is >103, whereas that of tiny spermatozoa is approximately 10–3 [3]. Currently, little is known about the power output derived from waves in an environment with such a low Reynolds number. In this study, we show that hamster spermatozoa progress efficiently by producing effective and ineffective strokes. Therefore, spermatozoa represent a good model for elucidating the mechanism by which a waveform generates the power output in a low Reynolds number environment.

FOOTNOTES

1 Correspondence: Fugaku Aoki, Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Shinryoiki-Seimei Building 302, Chiba 277-8562, Japan. FAX: 81 471 36 3698; aokif{at}k.u-tokyo.ac.jp Back

Received: 25 March 2005.

First decision: 24 April 2005.

Accepted: 16 August 2005.

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